Tumor resistance to chemotherapeutic agents resulting from the development of MultiDrug Resistance (MDR), remains a major cause of therapeutic failure and death. At least two proteins are well-known to play major roles in causing MDR, which is characterized by the resistance of tumor cells to a wide variety of agents of diverse structure and activity. Both proteins, P-glycoprotein (Pgp, product of the MDR1 gene) and the Multidrug resistance Related Protein (MRP, product of the MRP1 gene) are membrane transporters that produce increased drug effiux from tumor cells. Most of our understanding of the drug transporter systems comes from tissue culture studies in vitro. In the current application we propose to study the role of these transporters in drug uptake and efflux in human tumors maintained as xenografts in immuno-compromised mice. The project will test the hypotheses that overexpressing Pgp and/or MRP causes decreased drug accumulation and altered intracellular drug distribution in cancer cells and that inhibitors of Pgp and MRP reverse the action of the two transporters. Using a state-of-the-art two-photon microscope, we can study in real time how tumor cells in a living animal process anticancer drugs and respond to inhibitors of Pgp and MRP. These studies will provide direct in vivo information on the roles of Pgp and MRP in developing MDR and on the mechanisms of reversing MDR by inhibitors of Pgp and MRP in cancer cells. The proposal should lead to improved therapy for cancer patients whose tumors are resistant to anticancer agents. AIM 1 will use a two-photon microscope to clarify uptake, efflux and subcellular distribution of the highly fluorescent anticancer agents Adriamycin (ADR) and Mitoxantrone (MITOX) in human breast tumors maintained as xenografts in immuno-deficient mice. Wild type human vector only control breast tumors and tumors genetically engineered to express Pgp, MRP, or both Pgp and MRP will be studied for their ability to take up and effiux ADR and MITOX. AIM 2 will use a two-photon microscope to determine whether the inhibitors of the drug resistance transporters Pgp and MRP alter drug accumulation and retention in the wild type vector only control and resistant human breast tumors maintained as xenografts in nude nude mice. AIM 3 will use a two-photon microscope to determine the uptake and efflux of the inherently fluorescent antitumor agent MITOX in innately resistant human prostate tumors maintained as xenografts in immuno-deficient mice and will test whether modulators of Pgp and MRP reverse the resistance transporters in the prostate xenograft tumors.